Shear stress-exposed pulmonary artery endothelial cells fail to upregulate HSP70 in chronic thromboembolic pulmonary hypertension

Abstract

The pathophysiological mechanisms underlying chronic thromboembolic pulmonary hypertension (CTEPH) are still unclear. Endothelial cell (EC) remodeling is believed to contribute to this pulmonary disease triggered by thrombus and hemodynamic forces disbalance. Recently, we showed that HSP70 levels decrease by proatherogenic shear stress. Molecular chaperones play a major role in proteostasis in neurological, cancer and inflammatory/ infectious diseases. To shed light on microvascular responses in CTEPH, we characterized the expression of molecular chaperones and annexin A2, a component of the fibrinolytic system. There is no animal model that reproduces microvascular changes in CTEPH, and this fact led us to isolated endothelial cells from patients with CTEPH undergoing pulmonary endarterectomy (PEA). We exposed CTEPH-EC and control human pulmonary endothelial cells (HPAEC) to high- (15 dynes/cm2) or low- (5 dynes/cm2) shear stress. After high-magnitude shear stress HPAEC upregulated heat shock protein 70kDa (HSP70) and the HSP ER paralogs 78 and 94kDa glucose-regulated protein (GRP78 and 94), whereas CTEPH-ECs failed to exhibit this response. At static conditions, both HSP70 and HSP90 families in CTEPH-EC are decreased. Importantly, immunohistochemistry analysis showed that HSP70 expression was downregulated in vivo, and annexin A2 was upregulated. Interestingly, wound healing and angiogenesis assays revealed that HSP70 inhibition with VER-155008 further impaired CTEPH-EC migratory responses. These results implicate HSP70 as a novel master regulator of endothelial dysfunction in type 4 PH. Overall, we first show that global failure of HSP upregulation is a hallmark of CTEPH pathogenesis and propose HSP70 as a potential biomarker of this condition.

Fig 1. Characterization of endothelial cells from CTEPH patient.

(A). Representative image from PEA samples. (B) Images of HPAEC and CTEPH-EC in static condition and after exposition to low (5 dynes/cm²) and high shear stress (15 dynes/cm²). (C) Flow cytometry analyses of CD31 and CD90 expression in HPAEC and CTEPH-EC (n = 5). (D) Annexin V quantification of HPAEC and CTEPH-EC in static condition (n = 5).

Fig 2. Cytoplasmic protein expression in HPAEC and CTEPH-EC.

(A) Representative blot of HSP70 expression in static, low, and high magnitude shear stress (0, 5, and 15 dynes/cm²). Graphs are presented as the mean ± standard error. (*) HPAEC vs HPAEC 15, (#) CTEPH-EC vs CTEPH-EC 5, (&) HPAEC 15 vs CTEPH-EC 15, P < 0.05.(n = 6–7). (B) HSP90 (n = 6) and alpha 5 integrin (n = 4) expression in static condition. Data are presented as the mean ± standard error. (*) HPAEC vs CTEPH-EC, P < 0.05. All of the samples were normalized to ß-actin and run in the same gel.

Fig 3. Endoplasmic reticulum chaperones are downregulated in CTEPH-EC.

(A) Representative blot of the GRP94 expression in static, low, and high magnitude shear stress (0, 5, and 15 dynes/cm²). Data are presented as the mean ± standard error: (*) HPAEC vs CTEPH-EC, (&) HPAEC 15 vs CTEPH-EC 15. (n = 5–7). (B) GRP78 and PDI expression before and after shear stress exposure as described in methods. Data are presented as the mean ± standard error: (*) HPAEC vs HPAEC 15, (#) CTEPH-EC vs CTEPH-EC 5 (&) HPAEC 15 vs CTEPH-EC 15, P < 0.05. (n = 6–7). All of the samples were normalized to ß-actin and run in the same gel.

Fig 4. Expression of HSP70 and annexin A2 in the internal part of the pulmonary artery from CTEPH patients and the pulmonary artery from lung transplant donors (control).

(A) Immunohistochemistry analyses of HSP70 and (C) Annexin A2 expression. (B, D) Quantification of images from (A) and (C), respectively. Data are presented as the mean ± standard error. Control: n = 4 // Patients: n = 5. (*) Control vs CTEPH, P < 0.05.

Fig 5. Migration capacity of HPAEC and CTEPH-EC in basal and with HSP70 inhibitor VER-155008.

(A) Representative images of HPAECs and CTEPH-ECs basal and after VER-155008. (B) Quantification of the migration. Data are presented as the mean ± standard error: (*) HPAEC Basal vs HPAEC VER, (#) HPAEC Basal vs CTEPH-EC Basal, (&) CTEPH-EC Basal vs CTEPH-EC VER, P < 0.05. (n = 3).

Fig 6. Tube formation: Representative light microscopic images of the structures formed by HPAEC and CTEPH-EC seeded and incubated for 5h30 min in the absence or presence of 50 uM VER-155008 (n = 6).